The present invention relates to improved ligand-containing media, their method of preparation and use in the production of peptides, proteins, and the like, by chromatographic separation. In a preferred embodiment the invention comprises media having permanently attached via a covalent bond to an inert solid substrate an avidin polypeptide ligand in the dissociated renatured form which reversibly binds to certain molecules (proteins, peptides, nucleotides, oligonucleotides, and the like) and other molecules which bind to avidin via biotinylation or by way of their secondary/tertiary micromolecular structures.
The production of certain peptides and proteins for use in human health, animal health, industrial, food, and agricultural markets has been hampered by high cost and scarcity, particularly in the health care area. Peptides and proteins for human health uses are naturally synthesized by living organisms for their own needs and until recently, animals, plants, cadavers, serum and urine were the only sources from which these valuable biomolecules could be obtained. For example, porcine or bovine insulin was extracted from the pancreas of pigs or cattle for use by diabetics and HGH (human growth hormone) was obtained in small quantities from cadavers to treat infantile dwarfism. These biomolecules were usually obtained in very small quantities because only limited amounts were produced biologically or they were rapidly degraded by enzymes in their environment. Two new technologies have been developed which make possible the production of almost any peptide or protein in relatively large quantities: chemical and biological synthesis.
The chemical route is achieved by solid phase (Merrifield technique) and solution phase peptide synthesis; this approach is usually limited to peptides of less than 20 amino acid residues. Biological synthesis uses genetic engineering and recombinant DNA technologies and production of cells in tissue cultures or by microbial fermentation. The biological route has been the only practical approach to the production of higher molecular weight peptides in relatively large quantities.
Since interest in these biomolecules is based on their performance, purification of the desired biomolecule becomes a very important factor, especially in the health care and food additive industries, where the cost of purification alone, usually involving multiple process steps, can represent more than half of the total cost of producing the desired biomolecules.
The prior art has taught many techniques for covalently binding materials such as proteins to solid substrates as a technique for separating the bound species. For example, U.S. Pat. No. 4,732,811 (granted Mar. 22, 1988) describes the use of polymers containing polyaldehyde groups as capable of binding compounds containing primary amino groups (e.g., protein, antibodies and drugs).
Regardless of the synthetic route used, purification is necessary and liquid chromatography has been the universal tool used for these bioseparations. Among the chromatographic approaches available (ion exchange, size exclusion, reverse phase, hydrophobic interaction, and affinity), affinity chromatography has the potential for significantly reducing the number of purification steps required. Affinity columns based on avidin are known as being useful for the isolation of various biomolecules (D. A. Fuccillo, Biotechniques, 3 (6), 494-501 (1985)). In particular, avidin-biotin interactions have been applied to the isolation of proteins from biological synthetic routes. Avidin is a basic high-molecular weight glycoprotein found in egg whites; biotin is a low-molecular weight molecule with a fused imidazole-thiophene ring system which acts as a tag for recognition by avidin, resulting in an extremely stable avidin-biotin complex. Since the synthetic route chosen usually requires isolation of the desired biomolecule from very low concentrations in its environment, the extremely high affinity of avidin for biotin has been exploited in the chromatographic concentration and isolation of biotin-tagged ("biotinylated") molecules by use of avidin affinity columns. The specificity and affinity of avidin (native tetrameric form of 4 identical subunits) for biotin is extremely high and chromatography columns based on this principle have been used for analytical purposes. However, proteins and peptides containing the biotin-tag can not be recovered from an avidin tetramer column without using harsh conditions which invariably destroy the very biomolecule being isolated. Attempts to overcome the strong binding of biotin by avidin without losing the high specificity for binding biotinylated molecules have concentrated on using solid supports to stabilize the dissociated form of avidin; however, these columns have not been satisfactory for preparative use because of the presence of several classes of binding sites with less than desirable binding capacities as well as other deficiencies associated with the particular solid support matrices used (KP Henrikson, et al., Analytical Biochemistry, 94,366-370 (1979)).
Various routes have been used to anchor the avidin moiety to a solid support. Most commonly, agarose activated to a proper form for covalent coupling of the primary amino groups of avidin is used as a support (A. D. Landman, et al., J. Chem. Educ., 53 (9), 591 (1976)). However, these covalent linkages have not proven satisfactory due to chemical instability and resultant leaching of the avidin from the support, thus reducing the operational life of the column and also contaminating the purified product sought. Other major disadvantages of these particular columns include nonspecific adsorption of proteins, compressibility of the column matrix at high liquid flow rates resulting in back pressure and reduced flow, and the sensitivity of agarose to microbial degradation. In addition, agarose materials are not susceptible to easy cleaning and sterilization. Other supports based on polystyrene or silica (Japanese Kokai Patent Application JP 64-003129 A) have been used, but these suffer from even lower binding capacities than does agarose as well as incompatibility with certain biologically important ions.
For these reasons there is a need for a purification medium which allows efficient separation and subsequent isolation of biologically important molecules in a form satisfactory for the critical needs of such fields as health care and food additives.
The terms "adsorption" or "chromatographic adsorption" are intended in the specification and the appended claims in a broad, but perhaps not entirely pure technical, sense to embrace any form of binding between chemical species, other than covalent binding. Thus, binding by affinity or Van der Walls forces, while technically distinguishable, are both intended to be included within the broad meaning of "adsorption" as used herein.